α-Glucan is an α-D-glucose polymer. Various forms of α-glucan occur in nature. Among α-glucans, typical examples are glycogen and starch. However, glycogen and starch are significantly different from each other in structural and physical features.
Glycogen is the major storage polysaccharide in animals, fungi, yeasts and bacteria. Glycogen is water-soluble and forms a milky white solution. The molecular structure of glycogen in animals is well studied. Native glycogen is a homoglucan wherein a saccharide chain of grape sugars (glucoses) bonded linearly via α-1,4-glucosidic bond is branched via α-1,6-glucosidic bonds and the resulting branch is further branched to form a network structure. Native glycogen is composed of α-1,4-glucoside-linked chains having an average degree of polymerization of about 10 to about 14 and binding via α-1,6-glucosidic bonds. The molecular weight of native glycogen is described variously, and is estimated to be about 105 to about 108. Native glycogen occurs as a particle having a molecular weight of about 107 (β-particle) or as a larger particle (α-particle) formed by aggregation of β-particles. It is considered that the structure of glycogen in bacteria is similar to the structure of glycogen in animals. A glucan similar in structure to glycogen occurs in certain plants (for example, sweet corn) and is called vegetable glycogen (phytoglycogen).
Starch is the major storage polysaccharide in plants and occurs as a water-insoluble particle. This particle contains two different polysaccharides. The two polysaccharides are amylose and amylopectin. Amylose is composed of substantially linear D-glucose units linked with α-1,4 bonds. Amylopectin is a branched polymer considered to have a cluster structure. Each cluster unit is composed of α-1,4-linked glucosyl chains having an average degree of polymerization of about 12 to about 24 and binding to one another via α-1,6-glucosidic bonds. The cluster unit is further linked with a longer α-1,4-linked glucosyl chain having an average degree of polymerization of about 30 to about 100. The average chain length of the whole amylopectin is about 18 to about 25 in terms of a degree of polymerization. Starch amylopectin, similar to glycogen, is glucan bound via α-1,4-glucosidic bonds and α-1,6-glucosidic bonds, but glycogen is branched at a higher degree than amylopectin.
Recently, glycogen was proven to have an immunostimulating effect. Accordingly, glycogen can be expected for use as an immunostimulant, health food material and the like. In addition, the application of glycogen as a cosmetic material, a food material (flavoring material), and other industrial materials can be expected. Glycogen is utilized in various industrial fields. Applications of glycogen include, for example, a therapeutic agent for microbial infections, a humectant (for example, a cosmetic effective for improving the moisture retention of skin, a cosmetic for prevention of roughening of lips), a complex seasoning (for example, a complex seasoning having the taste of the eye of a scallop), an antitumor agent, an accelerator for formation of fermented milk, a colloid particle aggregate, a substance improving abrasion resistance of a hair surface, which influences ease in combing and luster of hair, a cellular stimulant (an epidermal cell stimulant, a fibroblast growth stimulant, and the like), an ATP production accelerator, an agent for ameliorating skin aging symptoms such as wrinkles, an agent for ameliorating skin roughening, a surface treatment agent for fluorescent particle, and a substrate in the synthesis of cyclic tetrasaccharide (CTS; cyclo{→6}-α-D-glcp-(1→3)-α-D-glcp-(1→6)-α-D-glcp-(1→3)-α-D-glcp-(1→}). Glycogen can be used in external preparations for skin (for example, skin lotion, emulsion, cream, essence, hair-growth medicine, hair growth tonic, mask, lip stick, lip cream, makeup base lotion, makeup base cream, foundation, eye color, cheek color, shampoo, rinse, hair liquid, hair tonic, permanent wave agent, hair color, treatment, bath agent, hand cream, leg cream, neck cream, body lotion, and the like), in a solution for eyes or the like.
Glycogen derived from mussels (moule) and vegetable glycogen (phytoglycogen) derived from sweet corn are marketed but is expensive and used mainly as a humectant in cosmetics. As a reagent, glycogen derived from various kinds of shellfish or animal livers is also marketed but extremely expensive and hardly industrially applicable.
Accordingly, it is desired to provide glycogen inexpensively in a large amount.
A branching enzyme (systematic name: 1,4-α-D-glucan:1,4-α-D-glucan 6-α-D-(1,4-α-D-glucano)-transferase, EC2.4.1.18, which is also referred to in this specification as BE) is an enzyme which cleaves α-1,4-glucosidic bonds and transfers the bond to an OH group at the 6-position of another glucosyl residue to form α-1,6-glucosidic bond. BE is distributed widely in animals, plants, mould fungi, yeasts and bacteria and catalyzes synthesis of a branched bond of glycogen or starch.
The catalytic action of potato-derived BE was examined in detail in the 1970's, and BE has been proven to catalyze an intermolecular branching reaction (FIG. 1A). It was proven in the late 1990's that BE catalyzes a cyclization reaction (FIG. 1B). By proving this cyclization reaction, it was logically estimated that an intramolecular branching reaction (FIG. 1C) is also catalyzed by BE. This is because from the microscopic viewpoint of cleavage of α-1,4-glucosidic bonds, transfer of the bond to an OH group at the 6-position of another glucosyl residue, and formation of α-1,6-glucosidic bond, these 3 reactions can be said to be identical. BE is regarded as one member of the glycoside hydrolase 13 family (α-amylase family) and considered to catalyze, at a single active center, cleavage of α-1,4-glucosidic bonds and transfer of the bond to an OH group at the 6-position by basically the same mechanism as that of α-amylase.
It is known that glycogen similar in structure and properties to native glycogen can be synthesized by allowing BE, together with another enzyme, α-glucan phosphorylase, to act on glucose-1-phosphate and oligosaccharide, or by allowing BE, together with glycogen synthase (or starch synthase), to act on UDP-glucose (or ADP-glucose). However, α-glucan phosphorylase is marketed as a reagent, but is extremely expensive. Further, acquisition of glycogen synthase and starch synthase is difficult. Glucose-1-phosphate, UDP-glucose, and ADP-glucose are extremely expensive. Accordingly, the problem of providing glycogen inexpensively in a large amount could not be solved by this method.
A macromolecule such as glucan is generally not a uniform molecule but a mixture of molecules having various sizes, and thus its molecular weight is evaluated in terms of a number-average molecular weight (Mn) or a weight-average molecular weight (Mw). The Mn is determined by dividing the total mass of the system by the number of molecules contained in the system. That is, the Mn is an average by number fraction. On the other hand, the Mw is an average by weight fraction. Given a completely homogeneous material, Mw=Mn, but a macromolecule generally has a molecular weight distribution, and therefore Mw>Mn. It follows that as Mw/Mn exceeds 1 and becomes higher, a degree of heterogeneity of the molecular weight becomes higher (that is, the molecular weight distribution is broader).
Amylose synthesized using an enzyme (for example, enzymatically synthesized amylose manufactured by Ajinoki Co., Ltd.) is known to have a narrow molecular weight distribution (the Mw/Mn <1.2 in Nonpatent Document 4; and the Mw/Mn=1.005 to 1.006 in Fujii, K. et al. (2003) Biocatalysis and Biotransformation, Vol. 21, pp. 167-172). On the other hand, amylose extracted from nature has a relatively broader molecular weight distribution, and the Mw/Mn is about 2 to about 5 (described in pp. 347-429 in Carbohydrates in food, edited by Eliasson, A.-C., Marcel Dekker, Inc., New York (1996); a degree of polymerization DP (number-average DPn, weight-average DPw) in Table 15 in Hizukuri, S., Starch: analytical aspects. By multiplying these DP by 162, the respective average molecular weights are given).
The Mn can be determined by evaluating the number of molecules. That is, the Mn of amylose or the like can be determined by measuring the number of reducing termini. The number of reducing termini can be determined, for example, by a modified Park-Johnson method described in Nonpatent Document 7. The Mn can also be determined for example by gel filtration chromatography (MALLS method) of using a differential refractometer in combination with a multi-angle laser-light scattering detector as described in Nonpatent Document 8. The Mw can be determined by the MALLS method described in Nonpatent Document 8.
In this specification, the molecular weight of a substrate is evaluated mainly in terms of the number-average molecular weight (Mn), while the molecular weight of produced glucan is evaluated mainly in terms of weight-average molecular weight (Mw). This is because when the product undergoes the cyclization reaction shown in FIG. 1B, Mn cannot be correctly evaluated by the method of evaluating the number of reducing termini, also because when the molecular weight of a very large molecule is evaluated, the number of reducing termini is relatively low, thus making accurate measurement of Mn difficult, and further, because the method of evaluating Mn by the MALLS method is based on the premise that fractionation by gel filtration is complete, so when the fractionation is incomplete, accurate evaluation of Mn is not feasible.
There are examples where BE is allowed to act on amylopectin or starch to give high molecular weight α-glucan. There are a large number of examples where BE alone is allowed to act on α-glucan (for example, amylose). However, there is no example where BE is allowed to act on amylose to give high molecular weight α-glucan having a molecular weight of about 1,000,000 or more. High molecular weight α-glucan obtained by allowing BE to act on amylopectin is considered to have increased branches on a fundamental structure of amylopectin, as shown in Nonpatent Document 17, and it can be said that glycogen (having a globular structure) was not synthesized. For example, Nonpatent Documents 1 and 2 describe that Neurospora crassa-derived BE is allowed to act on amylopectin or amylose thereby converting them into a highly branched glycogen-like molecule consisting of unit chains of 6-glucose units. However, the term “glycogen-like” merely means that a degree of coloration of the molecule by iodine is similar to that of glycogen. Amylose used therein as the substrate has number-average degrees of polymerization of 15, 22 or 130, indicating an Mn of about 2430, about 3600 and about 21000, respectively. Particularly, Nonpatent Document 2 describes that N. crassa-derived BE can act on short-chain amylose having an average degree of polymerization of 15 or 22, and the minimum degree of polymerization in amylose on which the plant-derived BE can act is 30 to 40 or more. Nonpatent Document 2 also describes that N. crassa-derived BE was suggested to act on a glucose chain of 12 residues or more thereby effecting transfer reaction of hexasaccharide as the minimum unit. As it can be seen from FIGS. 1 and 2 in Nonpatent Document 1 and FIGS. 3 and 4 in Nonpatent Document 2, when N. crassa-derived BE was allowed to act on amylopectin and amylose, the molecular weights of such substrates did not change. Further, FIGS. 4 and 5 in Nonpatent Document 1 and FIGS. 5 and 6 in Nonpatent Document 2 show that molecules slightly greater and slightly smaller than the substrate molecule are obtained, and a significantly high-molecular weight product was not observed.
For example, Nonpatent Document 3 describes that when maize BE I was allowed to act on amylose having an average chain length of greater than 300, a delay of the elution time of the product in gel filtration occurred, and this delay is due to a change in shape, but not to a change in molecular weight.
For example, Nonpatent Document 4 describes that the molecular weight of an amylopectin-like molecule obtained by allowing BE (particularly, Q enzyme) to act on amylose is decreased as the reaction time is increased.
For example, Nonpatent Document 5 describes that when potato-derived BE (Q enzyme) is allowed to act on amylose having an Mw of 67600, a reaction product having an Mw of 33500 can be obtained.
For example, Nonpatent Document 6 describes that when potato-derived BE is allowed to act on amylose having an Mn of 200,000, glucan having an Mw of 22,000 can be obtained.
For example, Nonpatent Document 7 describes that when Bacillus stearothrmophilus-derived BE is allowed to act on enzymatically synthesized amylose having an Mw of 302,000, a cyclization reaction is occurred to reduce the molecular weight of them. It is noted that the enzymatically synthesized amylose used as the substrate is known to have a narrow molecular weight distribution. For example, an enzymatically synthesized amylose's Mw/Mn <1.2 according to Nonpatent Document 4, and an enzymatically synthesized amylose's Mw/Mn=1.005 to 1.006, according to Fujii, K. et al. (2003) Biocatalysis and Biotransformation, Vol. 21, pp. 167-172. An enzymatically synthesized amylose's Mw/Mn <1.1, according to a pamphlet of a manufacturer Ajinoki Co., Ltd. Accordingly, the approximate Mn of the enzymatically synthesized amylose used in this document is about 252,000 to 302,000. Therefore, the Mn of the enzymatically synthesized amylose can be approximately estimated by dividing Mw by 1.1.
For example, Nonpatent Document 8 describes that when Aquifex aeolicus-derived BE is allowed to act on α-glucan, cyclized glucan can be obtained. This means that glucan is degraded into lower-molecular-weight products, as is evident from FIG. 1B.
For example, Nonpatent Document 9 describes that when Bacillus cereus-derived BE was allowed to act on enzymatically synthesized amylose of various sizes, glucan of almost the same size was obtained from all enzymatically synthesized amylose (FIG. 5.8). Further, from FIG. 5.9 in this document, it is evident that no component with a molecular weight of greater than about 1,000,000 was detected. Further, from a reaction model in FIG. 5.13 in this document, formation of highly branched and high molecular weight α-glucan cannot be expected. As is evident from FIG. 1, both larger and smaller molecules than the original molecule are generated in the intermolecular branching reaction by BE (FIG. 1A); a molecule smaller than the original molecule is generated in the cyclization reaction (FIG. 1B); and in the intramolecular branching reaction (FIG. 1C), the molecular weight is not changed before and after there action. Because the mechanisms of these reactions are the same, it cannot be expected that the 3 reactions occur at significantly different frequencies. Actually, the result in FIG. 5.8 in Nonpatent Document 9 reveals that all 3 reactions are catalyzed with some difference depending on the molecular weight of the substrate, resulting in formation of glucan of the same size from amylose of any size. In order to obtain high molecular weight glucan having a molecular weight of 1,000,000 or more from amylose, the intermolecular branching reaction of (A) is needed to be catalyzed at an overwhelmingly higher frequency, and among the resulting molecules, greater molecules are needed to undergo the reaction in the direction of further continuing polymerization. This cannot be expected from the conventional catalytic mechanism of BE, and no results obtained which suggest this.
Patent Document 2 describes a method of producing glucan having a degree of polymerization in the range of 50 to 5000 having an internal branched cyclic structural moiety and an external branched structural moiety, which comprises allowing BE (particularly, a branching enzyme) to act on amylose, partially degraded starch, debranched starch, amylose enzymatically synthesized with phosphorylase, maltooligosaccharide, and the like. In this method, the substrate is cyclized and formed by BE into a lower-molecular-weight molecule thereby producing cyclic glucan having a degree of polymerization of 50 to 5000 and a maximum degree of polymerization of 10,000. In this method, the product is obtained by forming the substrate into a lower-molecular-weight molecule, and thus high molecular weight amylose is used as the substrate. This is evident from paragraph 0066 describing that amylose having a degree of polymerization of about 400 or more can be preferably used. The molecular weight of amylose having a degree of polymerization of 400 is about 65,000, and whether or not high molecular weight α-glucan can be obtained using low molecular weight amylose as the substrate is not evident from this patent publication.
As described above, so far, it is believed that when BE is allowed to act on amylose, the amylose is converted into a lower-molecular-weight molecule, or even if the molecular weight of a certain molecule may be increased, there are few molecules undergoing polymerization to increase the molecular weight, and the molecular weight of the products are hardly changed.
Further, it is reported that α-glucan obtained by allowing BE to act on amylose is different from glycogen in that the α-glucan is easily degraded with pullulanase (Nonpatent Documents 10 and 16). There is also a document describing that “glycogen” was obtained by allowing BE to act on amylose (for example, Nonpatent Document 18 (Walker et al., Eur. J. Biochem. (1971) Vol. 20, pp. 14-21)), but in this document, the molecular weight of the resulting glucan is not measured, nor is digestibility analyzed.
Further, there are many examples wherein BE is allowed to act on amylose in order to examine the properties of the enzyme (for example, Patent Document 3 and Nonpatent Documents 11 to 12). In none of these examples, however, is the molecular weight of the reaction product measured.
It is known that BE (particularly plant-derived BE) hardly acts on short-chain amylose. For example, Nonpatent Document 13 describes that BE hardly acts on amylose having a degree of polymerization of 40 or less (molecular weight of about 6480). This is possibly because BE requires substrate amylose to have a certain higher order structure, but amylose not having a certain length can not have such higher order structure (Nonpatent Document 14). Further, it is considered that such higher order structure is related to temperature, and when the temperature is high, amylose cannot have such higher order structure.
Bacterium-derived BE seems to act on a short substrate (Nonpatent Document 15), but its action is known to be weak (Nonpatent Document 9, FIG. 4.5).
From the foregoing, it cannot be expected that highly branched and high molecular weight glucan having a molecular weight of 1,000,000 or more can be synthesized from amylose as the substrate by BE, and still more, it cannot be expected that the digestibility of the high molecular weight glucan with pullulanase and α-amylase is low. Further, because of the low activity thereof on enzymatically synthesized amyloses having Mns of 4800 and 9,300 (about 7% and 12% activity as compared the maximum activity thereof when enzymatically synthesized amylose having an Mn 270,000 is used as a substrate. FIG. 4.5 in Nonpatent Document 9), advantages of using amylose having an Mn of 8,000 or less (particularly an Mn of 4,000 or less) as a substrate have not been contemplated.
Further, in the conventional methods of producing glycogen, there is also the problem that significantly high expenditure is necessary for obtaining high-purity glycogen because the contents of electrolytes and monosaccharides are high unless the product is highly purified. For example, in the method of producing glycogen by adding BE to sucrose phosphorylase and α-glucan phosphorylase, addition of about 10 mM phosphoric acid to the reaction solution is needed, and the resulting reaction product contains a large amount of fructose and a small amount of phosphoric acid (sucrose+phosphoric acid+oligosaccharides→α-glucan+fructose+phosphoric acid). In the method wherein GP is combined with BE, the product contains a larger amount of electrolyte (glucose-1-phosphate+oligosaccharide→α-glucan+phosphoric acid). This also applies to the method wherein glycogen synthase (GS) is combined with BE (ADP-glucose+oligosaccharides→α-glucan+ADP).
Even if glycogen is extracted from a natural product, the glycogen is contaminated with various substances such as proteins, lipids and other carbohydrates in addition to electrolytes, and thus there is a problem of significantly high expenditure in obtaining high-purity glycogen.    Patent Document 1: Japanese Laid-open Publication No. 2000-316581    Patent Document 2: Japanese Patent No. 3107358, claim 1, column 0066    Patent Document 3: Japanese Patent National Phase PCT Laid-Open Publication No. 2002-539822    Nonpatent Document 1: Matsumoto et al., J. Biochem, Vol. 107, 118-122 (1990) (FIG. 2)    Nonpatent Document 2: Matsumoto and Matsuda, “Denpun Kagaku” (Starch Science), Vol. 30, pp. 212-222 (1983) (FIGS. 3 & 4)    Nonpatent Document 3: Boyer et al., Starch/staerke 34 Nr. 3, S. 81-85 (1982) (Table 1, FIG. 2 and FIG. 3)    Nonpatent Document 4: Kitamura, Polymeric Materials Encyclopedia, Vol. 10, pp. 7915-7922 (Table 2)    Nonpatent Document 5: Praznik et al., Carbohydrate Research, 227 (1992) pp. 171-182    Nonpatent Document 6: Griffin and Victor, Biochemistry Vol. 7, No. 9, September 1968    Nonpatent Document 7: Takata, H. et al., Cyclization reaction catalyzed by branching enzyme. J. Bacteriol., 1996. 178: pp. 1600-1606    Nonpatent Document 8: Takata, H. et al., J. Appl. Glycosci., 2003. 50: pp. 15-20    Nonpatent Document 9: Hiroki Takata Thesis For A Doctorate (Kyoto University, JP) 1997 (Studies on Enzymes Involved in Glycogen Metabolism of Bacillus Species)    Nonpatent Document 10: Charles Boyer and Jack Preiss, Biochemistry 1977, Vol. 16, No. 16, pp. 3693-3699    Nonpatent Document 11: Shinohara, M. L. et al., Appl Microbiol Biotechnol, 2001. 57(5-6): pp. 653-9    Nonpatent Document 12: Takata, H. et al., Appl. Environ. Microbiol., 1994. 60: pp. 3096-3104    Nonpatent Document 13: Borovsky, D., Smith, E. E. and Whelan, W. J. (1976) Eur. J. Biochem. 62, 307-312    Nonpatent Document 14: Borovsky, D., Smith, E. E. and Whelan, W. J. (1975) FEBS Lett. 54, 201-205    Nonpatent Document 15: Okada et al., “Denpun Kagaku” (Starch Science), Vol. 30, pp. 223-230 (1983)    Nonpatent Document 16: Kitahata, S, and Okada, S. (1988) in Handbook of amylase and related enzymes. Their sources, isolation methods, properties and applications. (The Amylase Research Society of Japan ed), pp. 143-154, Pergamon Press, Oxford    Nonpatent Document 17: Kawabata et al. (2002) J. Appl. Glycosci. Vol. 49, No. 3, 273-279    Nonpatent Document 18: Walker et al., Eur. J. Biochem. (1971) Vol. 20, pp. 14-21